Method for acquiring information related to size of nucleic acids contained in viral capsids using nanopore device, nanopore device and acquisition apparatus used in the acquisition method

Abstract

To provide a method and the like for acquiring information related to the size of nucleic acids contained in viral capsids using a nanopore device.SOLUTION: A nanopore device includes: a substrate which has a first surface and a second surface; a nanopore which penetrates from the first surface to the second surface and through which a capsid passes; a first chamber member; and a second chamber member. The first chamber member forms a first chamber filled with a first electrolytic solution with a surface of the first surface that includes at least a first opening of the nanopore. The second chamber member forms a second chamber filled with a second electrolytic solution with a surface of the second surface that includes at least a second opening of the nanopore. An acquisition method includes: a capsid passage step in which the capsid contained in the first electrolytic solution or the second electrolytic solution passes through the nanopore; and an ion current measurement step in which a change in the ion current is measured when the capsid passes through the nanopore.SELECTED DRAWING: Figure 6

Description

Technical Field

[0001] The disclosure in the present application relates to a method for obtaining information related to the size of nucleic acids contained in virus-derived capsids using nanopore devices, an apparatus for obtaining information related to the size of nucleic acids contained in virus-derived capsids, and a nanopore device used in the obtaining apparatus.

Background Art

[0002] Gene therapy is known for treating diseases by repairing and correcting the defects of cells that have malfunctioned due to gene abnormalities. As an example of gene therapy, a method is known in which a retrovirus or the like incorporating a therapeutic gene is used as a vector and introduced into cells that have malfunctioned. In this method, since a therapeutic gene is artificially incorporated into the retrovirus, which is a vector, quality control is required when actually used for treatment.

[0003] By the way, a device that forms nanopores (through holes penetrating the substrate) in the substrate and measures the change in ionic current when a sample passes through the nanopores has attracted attention as a device that can be widely applied to sensing of bacteria, DNA, proteins, etc.

[0004] As a related technique, for example, it is known to analyze the shape distribution of exosomes by measuring the change in ionic current when exosomes pass through nanopores formed in a substrate (see Patent Document 1).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] As described in Patent Document 1 above, it is known that the shape and the like of a minute sample such as an exosome can be measured by using a nanopore device. However, from the viewpoint of quality control of a virus vector incorporating a therapeutic gene, a method using a nanopore device has not been known.

[0007] As a result of intensive studies, the present inventors newly found that (1) the size of a virus-derived capsid increases as the size of the encapsulated nucleic acid increases, (2) by using a nanopore device, a minute difference in the size of the capsid can be measured as a change amount of an ionic current, and (3) information on the size of the nucleic acid incorporated into the capsid is reflected in the change amount of the ionic current of a virus-derived capsid incorporating a therapeutic nucleic acid (hereinafter, the capsid incorporating a therapeutic nucleic acid may be referred to as a "virus preparation").

[0008] That is, the disclosure in the present application provides a method for acquiring information related to the size of a nucleic acid contained in a virus-derived capsid using a nanopore device, the nanopore device used in the acquisition method, and an acquisition apparatus.

Means for Solving the Problems

[0009] The disclosure in the present application provides a method for acquiring information related to the size of a nucleic acid contained in a virus-derived capsid using a nanopore device, the nanopore device used in the acquisition method, and an acquisition apparatus, as shown below.

[0010] (1) A method for acquiring information related to the size of a nucleic acid contained in a virus-derived capsid using a nanopore device, wherein the nanopore device includes a substrate having a first surface and a second surface, a nanopore that penetrates from the first surface toward the second surface and through which the capsid passes, a first chamber member, a second chamber member, and The first chamber member forms a first chamber filled with a first electrolyte solution with a surface including at least the first opening of the nanopore on the first surface. The second chamber member forms a second chamber filled with a second electrolyte solution with a surface including at least the second opening of the nanopore on the second surface. The acquisition method is as follows. A capsid passage step in which the capsid contained in the first electrolyte solution or the second electrolyte solution passes through the nanopore, and An ion current measurement step of measuring a change in the ion current when the capsid passes through the nanopore, and The capsid passage step is as follows. By applying a voltage to the first electrolyte solution filled in the first chamber and the second electrolyte solution filled in the second chamber, the capsid contained in the first chamber passes through the nanopore in the direction of the second chamber, or the capsid contained in the second chamber passes through the nanopore in the direction of the first chamber. Acquisition method. (2) The larger the nucleic acid size encapsulated by the capsid, the larger the size of the capsid. The acquisition method according to (1) above. (3) An analysis step is included following the measurement step. The analysis step analyzes the presence or absence of nucleic acid to be encapsulated in the capsid based on the amount of change in the ion current measured in the measurement step. The acquisition method according to (2) above. (4) An analysis step is included following the measurement step. The analysis step calculates the size of the nucleic acid encapsulated in the capsid based on the amount of change in the ion current measured in the measurement step. The acquisition method according to (2) above. (5) The thickness of the substrate is larger than the size of the capsid. The acquisition method according to any one of (1) to (4) above. (6) The size of the nanopore is 1.2 times or more the average particle diameter of the capsid. The acquisition method according to any one of (1) to (4) above. (7) A substance having a higher viscosity than water is added to the first electrolyte and / or the second electrolyte. The acquisition method according to any one of (1) to (4) above. (8) A nanopore device used in an acquisition device for information related to the size of nucleic acid contained in a virus-derived capsid, The nanopore device includes a substrate having a first surface and a second surface, a nanopore penetrating from the first surface toward the second surface through which the capsid passes, a first chamber member, a second chamber member, and includes The first chamber member forms a first chamber filled with a first electrolyte with at least the surface of the first opening of the nanopore on the first surface, The second chamber member forms a second chamber filled with a second electrolyte with at least the surface of the second opening of the nanopore on the second surface, The thickness of the substrate is larger than the size of the capsid. Nanopore device. (9) The size of the nanopore is 1.2 times or more the average particle diameter of the capsid. The nanopore device according to (8) above. (10) An acquisition device for information related to the size of nucleic acid contained in a virus-derived capsid, The acquisition device includes a nanopore device, a measurement unit, an analysis unit, and includes The nanopore device includes a substrate having a first surface and a second surface, a nanopore penetrating from the first surface toward the second surface through which the capsid passes, a first chamber member, a second chamber member, and includes The first chamber member forms a first chamber filled with a first electrolyte solution with a surface including at least the first opening of the nanopore on the first surface. The second chamber member forms a second chamber filled with a second electrolyte solution with a surface including at least the second opening of the nanopore on the second surface. The measuring unit measures a change in ionic current when the capsid passes through the nanopore. Based on the amount of change in the ionic current measured by the measuring unit, the analysis unit analyzes the presence or absence of nucleic acid to be encapsulated in the capsid, and / or calculates the size of the nucleic acid encapsulated in the capsid. An acquisition device. (11) The nanopore device is the nanopore device according to (8) or (9) above. The acquisition device according to (10) above.

Advantages of the Invention

[0011] By the method for obtaining information related to the size of the nucleic acid contained in the virus-derived capsid using the nanopore device disclosed in the present application, by measuring the amount of change in the ionic current caused by the change in the size of the capsid, information regarding the size of the nucleic acid encapsulated in the capsid can be obtained. Therefore, since the size information of the nucleic acid encapsulated in the capsid can be easily obtained, the information can be used for quality control of virus preparations and the like.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Modes for Carrying Out the Invention

[0013] Hereinafter, a method for acquiring information related to the size of nucleic acids contained in virus-derived capsids using a nanopore device (hereinafter, may be simply referred to as the "acquisition method"), an apparatus for acquiring information related to the size of nucleic acids contained in virus-derived capsids (hereinafter, may be simply referred to as the "acquisition apparatus"), and a nanopore device used in the acquisition apparatus (hereinafter, may be simply referred to as the "device") will be described in detail. In this specification, members having the same kind of functions are given the same or similar reference numerals. And for members with the same or similar reference numerals, repeated explanations may be omitted.

[0014] In addition, the positions, sizes, ranges, etc. of the respective components shown in the drawings may not represent the actual positions, sizes, ranges, etc. in order to facilitate understanding. Therefore, the disclosure in this application is not necessarily limited to the positions, sizes, ranges, etc. disclosed in the drawings.

[0015] In addition, in this specification, (1) A numerical range represented by using "~" means a range including the numerical values described before and after "~" as the lower limit value and the upper limit value, (2) For numerical values, numerical ranges, and qualitative expressions (for example, expressions such as "identical", "the same", etc.), they indicate numerical values, numerical ranges, and properties including generally acceptable errors in the relevant technical field, (3) When described as "substantially in the shape of 〇〇", it includes the exact shape of 〇〇 and shapes that are grasped as approximately in the shape of 〇〇, and is interpreted as such.

[0016] (Embodiment of Device 1) With reference to FIG. 1, Device 1 according to the embodiment will be described. FIG. 1 is a schematic cross-sectional view of Device 1 according to the embodiment.

[0017] Device 1 includes a substrate 2, a nanopore 3 formed in the substrate 2, a first chamber member 51, and a second chamber member 61. The substrate 2 has a first surface 21 and a second surface 22, and the nanopore 3 penetrates from the first surface 21 to the second surface 22 of the substrate 2. When implementing the acquisition method, the capsid S passes through the nanopore 3.

[0018] The first chamber member 51 forms a first chamber 5 filled with a first electrolyte with the surface of the first surface 21 including at least the first opening 31 of the nanopore 3. The second chamber member 61 forms a second chamber 6 filled with a second electrolyte with the surface of the second surface 22 including at least the second opening 32 of the nanopore 3.

[0019] As used herein, "capsid" refers to the protein coat of a virus strain that has lost or partially lost its viral replication and propagation capabilities, and which incorporates the nucleic acid introduced for treatment and serves as a carrier (vector) for efficiently introducing and expressing the nucleic acid into cells. Also, as used herein, "nucleic acid" refers to DNA and RNA. DNA and RNA may be single-stranded or double-stranded.

[0020] The capsid is not particularly limited as long as its size increases as the size of the encapsulated nucleic acid increases. Although not limited, examples of the virus from which the capsid is derived include enveloped viruses such as retroviruses, lentiviruses, herpesviruses, and Sendai viruses (viruses in which the capsid is mainly covered with an envelope composed of lipids), and non-enveloped viruses such as adenoviruses and adeno-associated viruses (AAV) (viruses without an envelope). Among them, AAV is used in gene therapy for the treatment of various diseases because it can infect many types of cells, has no pathogenicity to humans, and the virus particles are physically stable.

[0021] More specific examples of viruses include, although not limited to, DNA viruses such as herpesviruses, poxviruses, and hepadnaviruses as enveloped viruses, and RNA viruses such as flaviviruses, togaviruses, coronaviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, and retroviruses. Also, although not limited to, DNA viruses such as adenoviruses, adeno-associated viruses (AAV), and papillomaviruses, and RNA viruses such as picornaviruses, caliciviruses, noroviruses, and rotaviruses are examples of non-enveloped viruses.

[0022] In addition, in this specification, the virus from which the capsid is derived includes, in addition to the wild-type virus, inactivated viruses (e.g., inactivated vaccine antigens, etc.), virus-like particles (VLP) without genetic information, viruses that retain foreign genes used as vectors (also referred to as virus vectors), and the like.

[0023] The material for forming the substrate 2 is not particularly limited as long as it can form the nanopore 3 and can measure the change in the ionic current of the capsid passing through the nanopore 3. Examples of the material for forming the substrate 2 include insulating materials commonly used in the field of semiconductor manufacturing technology. Examples of the insulating materials include Si, Ge, Se, Te, GaAs, GaP, GaN, InSb, InP, SiN, etc. Further, the substrate 2 may be formed of a thin film called a solid membrane using materials such as SiN, SiO 2 , HfO 2 and the like, or may be formed in a sheet shape called a two-dimensional material using materials such as graphene, graphene oxide, molybdenum disulfide (MoS 2 ), boron nitride (BN), etc. Further, the substrate 2 may be formed using an artificial membrane or a naturally occurring membrane such as a lipid bilayer. Measurement devices using a lipid bilayer are described in, for example, Japanese Patent Application Laid-Open No. 2011-527191 and Japanese Patent Application Laid-Open No. 2020-000056. The matters described in Japanese Patent Application Laid-Open No. 2011-527191 and Japanese Patent Application Laid-Open No. 2020-000056 are incorporated herein by reference. Further, a commercially available product may be used as the measurement device using a lipid bilayer. Examples of commercially available products capable of nanopore analysis using a lipid bilayer include MinION, GridION X5, SmidgION, PromethION, etc. manufactured by Oxford Nanopore Technologies.

[0024] Note that, for example, when a solid membrane or a two-dimensional material is used as the substrate 2 such that graphene can be formed on a substrate 2 with a film thickness of 1 nm or less, the film thickness can be made very thin. However, if the film thickness of the substrate 2 is very thin, it may be difficult to handle it without damage. Therefore, the substrate 2 may have a laminated structure in which a solid membrane or a two-dimensional material is laminated on a support plate formed of the above-described insulating material. In the case of a laminated structure, a solid membrane or a two-dimensional material may be laminated on a support plate having holes larger than the nanopores 3, and the nanopores 3 may be formed in the solid membrane or the two-dimensional material.

[0025] The thickness of the substrate 2 on which the nanopores 3 are formed is not particularly limited as long as the amount of change in the ionic current that varies depending on the size of the encapsulated nucleic acid can be measured when the capsid passes through the nanopores 3. In the technical field of measuring the ionic current when the object to be measured passes through the nanopores 3, generally, the smaller the volume of the nanopores 3, the more preferable. In other words, it is preferable that the substrate is thinner than the size of the object to be measured. The reason is that the thinner the substrate 2 is than the size of the object to be measured, the more various information when the object to be measured passes through the nanopores 3 is reflected in the change in the ionic current. As a result, the change in the measured ionic current reflects, for example, information such as the size information of the object to be measured and the orientation of the object to be measured when it enters the nanopores 3. From this viewpoint, the thickness of the substrate 2 may be 0.3 nm or more.

[0026] On the other hand, in the acquisition method disclosed in the present application, it is only necessary to be able to obtain the amount of change in the ionic current reflecting the size information of the encapsulated nucleic acid, in other words, the magnitude of the change in the ionic current when the capsid whose size has increased by encapsulating the nucleic acid passes through the nanopores 3. Therefore, in the device disclosed in the present application, the thickness of the substrate 2 may be smaller than the size of the capsid, but may also be larger. By making the thickness of the substrate 2 larger than the size of the capsid, the size information of the capsid is more strongly reflected in the change in the ionic current. Therefore, from the viewpoint of measuring the size of the capsid, the sensitivity can be increased by making the thickness of the substrate 2 larger than the size of the capsid. Also, the thicker the substrate 2 is, the more convenient the manufacturing and handling become.

[0027] Although the capsid varies depending on the type, it has a size (average particle diameter) of about 20 nm to about 100 nm. Therefore, the thickness of the substrate 2 is not limited, but examples include 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 110 nm or more, 120 nm or more, 130 nm or more. On the other hand, if the substrate 2 is made too thick with respect to the size of the capsid, the sensitivity decreases. Therefore, the upper limit of the thickness of the substrate 2 includes 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 275 nm or less, 250 nm or less, 225 nm or less, 200 nm or less, 190 nm or less, 180 nm or less, 170 nm or less, 160 nm or less, 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, 110 nm or less, 100 nm or less.

[0028] Also, the thickness of the substrate 2 may be defined in relation to the size of the capsid to be measured. Although not limited, when the size of the capsid is taken as 1, the lower limit of the thickness of the substrate 2 includes 0.015 times or more, and from the viewpoint of measuring the size information of the nucleic acid encapsulated in the capsid with higher sensitivity, 1.0 times or more, 1.2 times or more, 1.4 times or more, 1.6 times or more, 1.8 times or more, etc. On the other hand, the upper limit of the thickness of the substrate 2 includes 5 times or less, 4.5 times or less, 4 times or less, 3.5 times or less, 3 times or less, 2.75 times or less, 2.5 times or less, 2.25 times or less, 2 times or less.

[0029] The nanopore 3 is formed to penetrate the substrate 2 from the first surface 21 of the substrate 2 in the direction of the second surface 22 which is the opposite surface of the first surface 21. As described above, the device disclosed in the present application only needs to be able to obtain information regarding the size change of the capsid by encapsulating nucleic acids. Therefore, the size of the nanopore 3 may be appropriately adjusted to be larger than the capsid but not too large. Although not limited, when the size of the capsid is taken as 1, the lower limit values include 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2.0 times or more. On the other hand, the upper limit values include 4 times or less, 3.8 times or less, 3.6 times or less, 3.4 times or less, 3.2 times or less, 3.0 times or less, 2.8 times or less, 2.6 times or less, 2.4 times or less, 2.2 times or less.

[0030] Also, as described above, although the capsids vary depending on the type, they are about 20 nm to about 100 nm in size. Therefore, the lower limit values of the size of the nanopore 3 include 24 nm, 26 nm or more, 28 nm or more, 30 nm or more, 32 nm or more, 34 nm or more, 36 nm or more, 38 nm or more, 40 nm or more. On the other hand, the upper limit values of the nanopore 3 include 400 nm or less, 380 nm or less, 360 nm or less, 340 nm or less, 320 nm or less, 300 nm or less, 280 nm or less, 260 nm or less, 240 nm or less, 220 nm or less.

[0031] In addition, when the cross-sectional shape parallel to the first surface 21 of the nanopore 3 is circular, the size of the nanopore 3 means the diameter when described. When the cross-sectional shape parallel to the first surface 21 of the nanopore 3 is not circular, the size of the nanopore 3 means the diameter of the inscribed circle of the cross-section. When a material other than the lipid bilayer is used as the material of the substrate 2 for the nanopore 3, it may be formed by etching or the like as shown in the examples described later. Further, the nanopore 3 may be formed such that the first opening 31 of the nanopore 3 on the first surface 21 side and the second opening 32 of the nanopore 3 on the second surface 22 side have the same shape. Alternatively, the sizes of the first opening 31 and the second opening 32 may be different. For example, the nanopore 3 may be formed so as to spread from the first surface 21 to the second surface 22 in the substrate 2. In that case, the size of the nanopore 3 means the size of the first opening 31 (the size of the smaller opening) formed on the first surface 21.

[0032] In addition, although one example of the nanopore 3 formed on the substrate 2 is shown in FIG. 1, two or more nanopores 3 may be formed. When two or more nanopores 3 are formed on the substrate 2, the distance between adjacent nanopores 3 may be adjusted as necessary in order to improve the measurement accuracy of the capsid. Since how to set the distance between adjacent nanopores 3 is described in detail in International Publication No. 2020 / 138021, detailed description is omitted in the disclosure of the present application. The matters described in International Publication No. 2020 / 138021 are incorporated herein by reference.

[0033] The first chamber member 51 and the second chamber member 61 are preferably formed of an electrically and chemically inert material. Although not limited, examples of the material include, for example, glass, sapphire, ceramic, resin, rubber, elastomer, SiO 2 , SiN, Al 2 O 3 and the like.

[0034] The first chamber 5 and the second chamber 6 are formed so as to sandwich the nanopore 3, and there is no particular limitation as long as the capsid introduced into the first chamber 5 can move through the nanopore 3 to the second chamber 6, or the capsid introduced into the second chamber 6 can move through the nanopore 3 to the first chamber 5. For example, the first chamber member 51 and the second chamber member 61 may be separately manufactured and adhered to the substrate 2 to be liquid-tight. Alternatively, a substantially rectangular parallelepiped box member with one face open may be formed, the substrate 2 may be inserted and fixed at the center of the box, and then the open face may be sealed liquid-tight. In that case, the first chamber member 51 and the second chamber member 61 do not mean separate members, but mean a part of the box member divided by the substrate 2. Although not shown, holes for filling and discharging the electrolytic solution and the capsid solution, inserting electrodes and / or leads may be formed in the first chamber member 51 and the second chamber member 61 as necessary.

[0035] (Embodiment of the acquisition device 1a) Referring to FIG. 2, an embodiment of the acquisition device 1a will be described. FIG. 2 is a schematic cross-sectional view showing an example of a configuration example of the acquisition device 1a according to the embodiment.

[0036] The acquisition device 1a shown in FIG. 2 includes at least a measurement unit 7 and an analysis unit 8 in addition to the device 1 according to the embodiment. The device 1 has been described in the above "Embodiment of the device 1". Therefore, since it will be a duplicate description, a detailed description of the device 1 will be omitted.

[0037] In the example shown in FIG. 2, a first electrode 52 formed at a location in contact with the first electrolytic solution in the first chamber 5, a second electrode 62 formed at a location in contact with the second electrolytic solution in the second chamber 6, and a power source 54 for applying a voltage between the first electrode 52 and the second electrode 62 are shown. However, the first electrode 52, the second electrode 62, and the power source 54 may be prepared separately from the acquisition device 1a and attached to the acquisition device 1a when implementing the acquisition method. That is, the first electrode 52, the second electrode 62, and the power source 54 are optional additional configurations in the acquisition device 1a.

[0038] Further, the acquisition device 1a may optionally include a display unit 9 for displaying the result analyzed by the analysis unit 8, a program memory 10 storing a program for operating the analysis unit 8 and the display unit 9 in advance, and a control unit 11 for reading and executing the program stored in the program memory 10. The program may be stored in the program memory 10 in advance, or may be recorded on a recording medium and stored in the program memory 10 using an installation means.

[0039] The first electrode 52 and the second electrode 62 can be formed of known conductive metals such as aluminum, copper, platinum, gold, silver, silver / silver chloride, and titanium. FIG. 2 shows an example in which the first electrode 52 and the second electrode 62 are formed so as to sandwich the nanopore 3, and a voltage is applied so that a direct current flows with the first electrode 52 side as the negative electrode and the second electrode 62 side as the positive electrode. Alternatively, the first electrode 52 side may be the positive electrode and the second electrode 62 side may be the negative electrode. Depending on the charge of the capsid described later, which side of the first electrode 52 and the second electrode 62 is positive may be determined as appropriate.

[0040] The first electrode 52 is not particularly limited as long as it is formed at a location in contact with the first electrolyte in the first chamber 5. In the example shown in FIG. 2, the first electrode 52 is disposed on the inner surface of the first chamber member 51 via a lead 53. Alternatively, the first electrode 52 may be disposed on the first surface 21 of the substrate 2 or in the space in the first chamber 5 via a lead 53. Further alternatively, the first electrode 52 may be disposed so as to penetrate the first chamber member 51 through a hole formed in the first chamber member 51.

[0041] The second electrode 62 is not particularly limited as long as it is formed at a location in contact with the second electrolyte in the second chamber 6, similar to the first electrode 52. In the example shown in FIG. 2, the second electrode 62 is disposed on the inner surface of the second chamber member 61 via a lead 63. Alternatively, the second electrode 62 may be disposed on the second surface 22 of the substrate 2 or in the space within the second chamber 6 via a lead 63. Further alternatively, the second electrode 62 may be disposed so as to penetrate the second chamber member 61 through a hole formed in the second chamber member 61.

[0042] In the example shown in FIG. 2, the first electrode 52 is connected to a power source 54 and a ground 55 via a lead 53. The second electrode 62 is connected to a measurement unit 7 and a ground 64 via a lead 63. In the example shown in FIG. 2, the power source 54 is connected to the first electrode 52 side and the measurement unit 7 is connected to the second electrode 62 side. However, the power source 54 and the measurement unit 7 may be provided on the same electrode side.

[0043] The power source 54 is not particularly limited as long as it can pass a direct current through the first electrode 52 and the second electrode 62. The measurement unit 7 is not particularly limited as long as it can measure the ion current generated over time when the first electrode 52 and the second electrode 62 are energized. Although not shown in FIG. 2, the acquisition device 1a may include a noise removal circuit, a voltage stabilization circuit, etc. as necessary.

[0044] The measurement unit 7 is not particularly limited as long as it can measure the change amount of the ion current when the capsid S passes through the nanopore 3, and examples include known ammeters.

[0045] When the capsid S passes through the nanopore 3, the ion current flowing through the nanopore 3 is blocked by the capsid S, and the ion current flowing through the nanopore 3 changes. The analysis unit 8 analyzes the change amount of the ion current measured by the measurement unit 7. Therefore, by performing data analysis by the analysis unit 8 based on the measured change amount of the ion current, information regarding the size of the nucleic acid encapsulated in the capsid S can be analyzed. And, as described above, the size of the capsid S increases as the size of the encapsulated nucleic acid increases (as the nucleic acid becomes longer). Therefore, (1) measure in advance the change amount of the ion current when the capsid S before introducing the nucleic acid passes through the nanopore 3, (2) measure the change of the ion current when the capsid S, which is the measurement object, passes through the nanopore 3, and (3) by comparing the previously measured change amount of the ion current with the change amount of the ion current of the measurement object, (4) it is possible to analyze the presence or absence of the nucleic acid to be encapsulated in the capsid S, in other words, whether the intended nucleic acid has been introduced into the capsid S.

[0046] Also, (1) measure in advance the change amount of the ion current when the capsid S before introducing the nucleic acid and the capsid S encapsulating nucleic acids with different lengths pass through the nanopore 3, (2) measure the change of the ion current when the capsid S, which is the measurement object, passes through the nanopore 3, and (3) by comparing the previously measured change amount of the ion current with the change amount of the ion current of the measurement object, (4) it is possible to calculate the size of the nucleic acid encapsulated in the capsid.

[0047] The analysis unit 8 may be provided with a storage unit that stores data obtained by previously measuring (a) the change amount of the ion current when the capsid S before introducing the nucleic acid passes through the nanopore 3, or (b) the change amount of the ion current when the capsid S before introducing the nucleic acid and the capsid S encapsulating nucleic acids with different lengths pass through the nanopore 3. Also, when performing analysis by the analysis unit 8, known machine learning may be used for the analysis. By using machine learning, it can be expected that the analysis accuracy will be improved.

[0048] The display unit 9 only needs to be able to display the amount of change in the measured ion current and the results analyzed by the analysis unit 8, and a known display device such as a liquid crystal display, a plasma display, or an organic EL display may be used. The program memory 10 is not particularly limited as long as it can store a program for operating the analysis unit 8 and the display unit 9, and examples include ROMs such as mask ROM, PROM, EPROM, and EEPROM. The control unit 11 is not particularly limited as long as it can read and execute the program stored in the program memory 10, and examples include a processor (CPU) or a general-purpose computer equipped with a CPU.

[0049] Note that the above-described device 1 and acquisition device 1a are merely examples of the embodiments and are not limited. Within the scope of the technical idea disclosed in this application, any combination of the illustrated various embodiments and any forms selected from the optional additional matters may be used.

[0050] (Embodiment of the acquisition method) Referring to FIG. 3, an embodiment of the acquisition method will be described. FIG. 3 is a flowchart of the acquisition method according to the embodiment. The acquisition method according to the embodiment includes a capsid passage step (ST1) and an ion current measurement step (ST2) as essential steps. Although an analysis step (ST3) is described in FIG. 3, in the acquisition method according to the embodiment, the analysis step (ST3) is not an essential configuration but an optional additional configuration.

[0051] In the capsid passage step (ST1), by applying a voltage to the first electrolyte filled in the first chamber 5 and the second electrolyte filled in the second chamber 6, the capsid S contained in the first chamber 5 passes through the nanopore 3 in the direction of the second chamber 6, or the capsid S contained in the second chamber 6 passes through the nanopore 3 in the direction of the second chamber 6. The first electrolyte and the second electrolyte only need to be able to conduct electricity between the first electrode 52 and the second electrode 62, and a solution (electrolyte) containing ions such as TE buffer, PBS buffer, HEPES buffer, and aqueous KCl solution, which are known in the art, may be used.

[0052] In addition, when performing the capsid passage step, optionally, a substance having a higher viscosity than water may be added to increase the viscosity of the first electrolyte solution and / or the second electrolyte solution. By increasing the viscosity of the first electrolyte solution and / or the second electrolyte solution, the time for capsid S to pass through nanopore 3 can be lengthened. The viscosities of the first electrolyte solution and the second electrolyte solution may be the same or different as long as the time for capsid S to pass through nanopore 3 can be lengthened. When the viscosities of the first electrolyte solution and the second electrolyte solution are the same, the conditions received from the electrolyte solution before and after capsid S passes through nanopore 3 (for example, the resistance of the electrolyte solution received by nanopore 3) are the same. Therefore, the viscosities of the first electrolyte solution and the second electrolyte solution may be different, but it is preferable that the difference in viscosity is not too large. It is more preferable that the viscosities of the first electrolyte solution and the second electrolyte solution are the same.

[0053] Examples of the substance having a higher viscosity than water include glycerin, DMSO, polyethylene glycol, hydrogel, xanthan gum, etc. Although the viscosity of water varies with temperature, it is about 1 mP·s at about 20°C. Although not limited, examples of the lower limit of the viscosity of the first electrolyte solution and / or the second electrolyte solution after adding the substance are 2 mPa·s or more, 4 mPa·s or more, 6 mPa·s or more, 8 mPa·s or more, 10 mPa·s or more, 15 mPa·s or more, 20 mPa·s or more, 25 mPa·s or more, 30 mPa·s or more, 35 mPa·s or more, 40 mPa·s or more, 45 mPa·s or more, 50 mPa·s or more, etc. at about 20°C. On the other hand, examples of the upper limit are 1000 mPa·s or less, 900 mPa·s or less, 800 mPa·s or less, 700 mPa·s or less, 600 mPa·s or less, 500 mPa·s or less, 450 mPa·s or less, 400 mPa·s or less, 350 mPa·s or less, 300 mPa·s or less, 250 mPa·s or less, 200 mPa·s or less, 150 mPa·s or less, 100 mPa·s or less, etc. By increasing the viscosity of the first electrolyte solution and / or the second electrolyte solution, when performing the capsid passage step, the time for capsid S to pass through nanopore 3 becomes longer. Therefore, there is an effect that information regarding the size of capsid S (information regarding the size of the nucleic acid encapsulated in the capsid) can be obtained with higher accuracy.

[0054] When the first chamber 5 is already filled with the first electrolytic solution and the second chamber 6 is already filled with the second electrolytic solution, the capsid may be introduced into the first chamber 5 or the second chamber 6, and the capsid passing step (ST1) may be carried out.

[0055] When the first chamber 5 of the acquisition device 1a is not filled with the first electrolytic solution and the second chamber 6 is not filled with the second electrolytic solution, a preparation step may be carried out before carrying out the capsid passing step (ST1). The preparation step can be carried out according to the following procedure. (1) Fill the first chamber 5 with the first electrolytic solution and fill the second chamber 6 with the second electrolytic solution. A liquid junction can be established between the inside of the first chamber 5 and the inside of the second chamber 6 through the nanopore 3. (2) Introduce the capsid S into the first chamber 5 or the second chamber 6. Note that the procedures described in (1) and (2) above may be carried out separately, but the electrolytic solution already containing the capsid S may also be introduced into the first chamber 5 or the second chamber 6.

[0056] In the capsid passing step (ST1) shown in FIG. 3, by energizing the first electrode 52 disposed in the first chamber 5 and the second electrode 62 disposed in the second chamber 6, in addition to normal diffusion, the capsid S passes through the nanopore 3 formed in the substrate 2 by electrophoresis.

[0057] In the ion current measurement step (ST2), the change in the ion current generated by energization is measured over time by the measurement unit 7. Therefore, when the capsid S passes through the nanopore 3, a large change in the ion current corresponding to the size of the capsid S can be measured.

[0058] The amount of change in the ion current obtained by the acquisition method according to the embodiment includes information regarding the size of the capsid S. And the information regarding the size of the capsid S includes information regarding the size of the nucleic acid encapsulated by the capsid S. Therefore, the acquisition method disclosed in the present application has the following effects.

[0059] (1) By measuring the size of the capsid, information regarding the size of the nucleic acid encapsulated within the capsid can be obtained. Therefore, information regarding the size of the encapsulated nucleic acid can be obtained without destroying the capsid. (2) When manufacturing a viral preparation, if the target nucleic acid is not contained in the capsid, or if the target nucleic acid is contained in the capsid in a cleaved state, the manufactured viral preparation will not substantially achieve a therapeutic effect, and moreover, there are concerns about side effects caused by the immune response. Information regarding the size of the nucleic acid encapsulated within the capsid is very useful for examining whether a viral preparation used in gene therapy is correctly manufactured. Therefore, by the acquisition method of the present application, the quality control of the viral preparation can be performed non-destructively. (3) Since the acquisition method disclosed in the present application can measure the ion current for each capsid, a large number of samples are not required. Therefore, in addition to the quality control after the manufacture of the viral preparation, it is also useful for in-process sampling checks and the like.

[0060] Subsequently, in the acquisition method, the analysis step (ST3), which is an optional additional configuration, will be described. In the analysis step (ST3), information on the nucleic acid encapsulated within the capsid is analyzed from the amount of change in the ion current measured in the ion current measurement step (ST2). The content to be analyzed is the same as that of the analysis unit 8 in the "Embodiment of the Acquisition Device 1a" above, and includes (a) analyzing the presence or absence of the nucleic acid to be encapsulated in the capsid S, in other words, whether the nucleic acid has been introduced into the capsid S, and / or (b) calculating the size of the nucleic acid encapsulated within the capsid.

[0061] Note that the analysis step (ST3) may be performed manually or processed using a computer or the like. For example, when performing manual analysis, based on the amount of change in ion current (the difference between the baseline of the ion current and the peak value of the amount of change in the ion current), (a) the amount of change in the ion current of the capsid not containing nucleic acid and the amount of change in the ion current of the capsid containing nucleic acid are referred to, and it may be analyzed whether nucleic acid has been introduced into the capsid S to be measured. Also, (b) the amount of change in the ion current when the capsid S before introducing nucleic acid and the capsid S containing nucleic acids of different lengths pass through the nanopore 3 is measured in advance, and the length of the nucleic acid encapsulated and the amount of change in the ion current are graphed based on the measured results. The length of the nucleic acid contained in the capsid S may be calculated by referring to the graph of the amount of change in the ion current of the capsid to be measured.

[0062] When analyzing with a computer or the like, the above analysis may be automated. Also, the accuracy of the analysis may be improved by using machine learning or the like.

[0063] When the acquisition method according to the embodiment has the analysis step (ST3), in addition to the effects described in the above (1) to (3), the following effects are achieved. (4) By performing the analysis step, the presence or absence of nucleic acid encapsulated in the capsid and the length of the nucleic acid contained can be grasped more accurately. Therefore, when used for quality control of virus preparations, quality control can be performed in more detail. In particular, by measuring in advance the amount of change in the ion current when the capsid S containing nucleic acids of different lengths passes through the nanopore 3, it is possible to grasp how the nucleic acid to be introduced is cleaved, enabling feedback to the manufacturing process and the like.

[0064] Note that the above-described acquisition device 1a also exhibits the effects described in the above (1) to (4) achieved by the acquisition method. And the device 1 exhibits the effect that it can be used for the acquisition method and the acquisition device 1a that exhibit the effects described in the above (1) to (4).

[0065] Examples are given below to specifically describe the embodiments disclosed in this application. However, these examples are merely for the purpose of explaining the embodiments and do not represent any limitation or restriction of the scope disclosed in this application.

Example

[0066] <Example 1> 〔Fabrication of Device 1〕 A 4-inch silicon wafer coated on both sides with a 50-nm-thick SiNx layer was diced into 30 mm × 30 mm chips. The silicon layer was partially dissolved by wet etching in an aqueous KOH solution to form a 30-nm-thick SiNx film. Then, an electron beam resist (ZEP520A, zeon) was spin-coated on the SiNx film, pre-baked at 180 °C using a hot plate, and then a circular pattern was drawn by electron beam lithography and developed. Using the resulting residual resist layer as a mask, a 66-nm-diameter nanopore was opened by reactive ion etching with CHF 3 etching gas. Finally, the nanopore chip with nanopores formed on the SiNx substrate was fabricated by immersing it in N,N-dimethylformamide overnight and washing it with ethanol / acetone.

[0067] Next, one surface of the fabricated nanopore chip was coated with a polyimide layer. This is to reduce the capacitance of the nanopore chip and reduce noise. More specifically, an imide precursor of photosensitive polyimide (PN-2010, Toray Industries, Inc.) was spin-coated on the nanopore film. After baking, ultraviolet light was irradiated by LED lithography and developed to dissolve the polyimide with a diameter and thickness of 5 μm around the nanopores.

[0068] The fabricated nanopore chip was sealed with two polymer blocks (a first chamber member and a second chamber member) made of polydimethylsiloxane (PDMS) to fabricate a first chamber and a second chamber. These blocks were fabricated by polymerizing a PDMS precursor (Sylgard 184, Dow) at 80 °C on an SU-8 mold. The mold had an I-shaped pattern with a submillimeter width and height to form trenches on the polymer block that functioned as channels for flowing the capsid solution into the nanopores. Before sealing, three holes were punched in the blocks. Subsequently, after subjecting the nanopore chip and the polymer blocks (the first chamber member and the second chamber member) to oxygen plasma for surface activation, the nanopore chip and the polymer blocks were joined to fabricate Device 1.

[0069] [Fabrication of Acquisition Device 1a for Implementing the Acquisition Method] Ag / AgCl rods were used as the first electrode and the second electrode and inserted into the first chamber and the second chamber through the holes on both sides of the polymer block. The ionic current passing through the nanopore was measured by first amplifying the output current passing through one of the rods using a custom-designed amplifier, then digitizing it using a high-speed digitizer (PXI-5922, NI), and accumulating it in a solid-state drive (PXI-8267, NI) at a sampling rate of 1 MHz under the applied voltage Vb.

[0070] [Implementation of the Acquisition Method] [Example 2] (1) Fabrication of AAV Empty Capsids 293EB cell line expressing adenovirus E1a, adenovirus E1b, and Bcl-xL (Tomono T., et al., “Highly efficient ultracentrifugation-free chromatographic purification of recombinant AAV serotype 9”, Mol. Ther. Methods Clin. Dev. 11, 180-190 (2018)) was seeded at a density of 40,000 cells / cm into a 550 mL multi-stage flask (HYPERFlask, Corning, Corning, NY, USA) supplemented with Dulbecco's Modified Eagle Medium (DMEM high glucose, FUJIFILM Wako, Osaka, Japan) and 10% fetal bovine serum (Thermo Fisher, Waltham, MA, USA), and cultured for 3 days. Then, pCAX (CAG promoter backbone plasmid for generating empty capsids) (32.5 μg / flask) (Takara Bio, Kusatsu, Shiga, Japan), pR2C8 (serotype 8) or pR2C9 (serotype 9) (32.5 μg / flask), and helper plasmid (65 μg / flask) in DMEM (Nacalai Tesque, Nakagyo-ku, Kyoto) containing 2 mM L-alanyl-L-glutamine solution (100x) were used for transfection with polyethyleneimine max (520 μg / flask) (Polysciences, Warrington, PA, USA). Ten days after transfection, the culture supernatant was collected and treated with endonuclease (Kaneka, Minato-ku, Tokyo, Japan) at 18.5 U / mL and MgCl 2 (Nacalai Tesque) at 5 mM for 30 minutes at 37°C. Next, rAAV was purified using the AAVpro® Concentrator kit (Takara, Japan) or PhyTip® column CaptureSelect® AAVX (PhyNexus, USA). 2 (Nacalai Tesque) at 5 mM for 30 minutes at 37°C. Next, rAAV was purified using the AAVpro® Concentrator kit (Takara, Japan) or PhyTip® column CaptureSelect® AAVX (PhyNexus, USA).

[0071] The rAAV genome copy number was analyzed using the AAVpro® Titration Kit (for real-time PCR) Ver. 2 (Takara, Japan) on a QuantStudio 3 real-time PCR system (Applied Biosystems, Waltham, MA, USA).

[0072] (2) Preparation of AAV Capsids Encapsulating Nucleic Acids A 550 mL multi-stage flask (HYPERFlask, Corning, Corning, NY, USA) supplemented with Dulbecco's Modified Eagle Medium (DMEM high glucose, FUJIFILM Wako, Osaka, Japan) and 10% fetal bovine serum (Thermo Fisher, Waltham, MA, USA) was seeded with 293EB cell line expressing adenovirus E1a, adenovirus E1b, and Bcl-xL at a density of 40,000 cells / cm 2 . The cells were cultured for 3 days. Then, pAAV-ZsGreen1 (2599 bases, manufactured by Takara Bio Inc. #6231) or pAAV-ZsGreen1-short (1451 bases, pAAV-ZsGreen1 digested with restriction enzymes) (32.5 μg / flask), pR2C8 (serotype 8) or pR2C9 (serotype 9) (32.5 μg / flask), and a helper plasmid (65 μg / flask) were used in DMEM (Nacalai Tesque, Nakagyo-ku, Kyoto) containing 2 mM L-alanyl-L-glutamine solution (100x) for transfection with polyethyleneimine max (520 μg / flask) (Polysciences, Warrington, PA, USA). Ten days after transfection, the culture supernatant was collected and treated with endonuclease (Kaneka, Minato-ku, Tokyo, Japan) at 18.5 U / mL and MgCl 2 (Nacalai Tesque) at 5 mM for 30 minutes at 37°C. Next, rAAV was purified using the AAVpro® Concentrator Kit (Takara, Japan) or PhyTip® Column CaptureSelect® AAVX (PhyNexus, USA).

[0073] (3) Measurement Conditions For the first electrolyte solution and the second electrolyte solution, 1.37 M NaCl (manufactured by Nippon Gene Co., Ltd.: 10×PBS Buffer(-), model number: 314-90185) was used. The first electrolyte solution was filled into the first chamber through the holes formed in the block. Also, the capsids encapsulating nucleic acids were filled into the first chamber. The second electrolyte solution was filled into the second chamber through the holes formed in the block. A voltage of 0.3 V was applied so that the first electrode 52 was the positive electrode and the second electrode 62 was the negative electrode, and the ionic current Iion was measured.

[0074] Figure 4 shows the changes in the ionic current when empty capsids without nucleic acids (displayed as "AAV9 / empty"), capsids encapsulating 1451-base DNA (displayed as "AAV9 / 1.5 kb DNA"), and capsids encapsulating 2599-base DNA (displayed as "AAV9 / 2.6 kb DNA") passed through nanopore 3. The graph in Figure 4 is the result of averaging 100 raw data points measured at 1 MHz (adjacent averaging).

[0075] FIG. 5 is a plot of Ip, which is the amount of change in the ionic current when individual capsids pass through the nanopore. Note that the display of "AAV9 / 1.5kb" is omitted in FIG. 5. In the example shown in FIG. 5, the amount of change in the ionic current (Ip) is defined as the difference between the baseline (base), which is the value of the ionic current measured when the capsid has not entered nanopore 3, and the average value (Av) of the ionic current values during the time td (the time from when the ionic current has dropped sharply from the baseline until just before it starts to rise sharply back to the baseline) when the entire capsid is passing through the nanopore, but is not limited to this definition. The amount of change in the ionic current (Ip) may be defined otherwise as long as the size information of the nucleic acid encapsulated in the capsid is reflected. For example, the amount of change in the ionic current (Ip) may be defined as the difference between the average value of the minimum values of a plurality of peaks measured during the td period (the lower side of the waveform during the td period shown in FIG. 5) and the baseline. Alternatively, it may be defined as the difference between the average value of the maximum values of a plurality of peaks measured during the td period (the upper side of the waveform during the td period shown in FIG. 5) and the baseline. FIG. 6 shows the average value of the amount of change in the ionic current Ip obtained from the plot in FIG. 5, which was 5.1 nA when the encapsulated DNA was 0, 6.1 nA when the encapsulated DNA was 1451 bases, and 6.7 nA when the encapsulated DNA was 2599 bases.

[0076] As shown in Fig. 6, since the amount of change in the ionic current measured by the size of the encapsulated nucleic acid was different, the size of the capsid was examined. The results are shown in Fig. 7. More specifically, empty capsids (Empty) and capsids encapsulating 2,599-base DNA (Full) were photographed with a transmission electron microscope (Figs. 7a and b). As shown by the arrows in Figs. 7a and b, the vector diameter (dvec) indicated by the arrow for each capsid was measured using ImageJ software (scale bar is 20 nm). Fig. 7c shows the distribution of dvec obtained from 2,052 Empty images and 1,576 Full images. The average diameter of Empty was 22.88 nm, and the average diameter of Full was 25.84 nm, with a size difference Δdvec of about 3 nm between the two (p < 0.0001). From the above results, the size of the capsid increased as the size of the encapsulated nucleic acid increased, and as a result, a difference occurred in the amount of change in the measured ionic current. Therefore, it was confirmed that the difference in the amount of change in the ionic current reflected the size information of the encapsulated nucleic acid.

[0077] Fig. 8 shows a graph with the length of the encapsulated nucleic acid on the horizontal axis and the average value of the measured amount of change in the ionic current (Ip) on the vertical axis. Note that the amount of change in the ionic current (Ip) shown in Fig. 8 is the difference between the average value of the minimum values of a plurality of peaks measured during the td period and the baseline, which is different from Figs. 5 and 6. In the example shown in Fig. 8, the number of plots is 3 points, but by increasing the number of plots, it is possible to create a more accurate approximation curve. By creating a graph like the one shown in Fig. 8 in advance, the length of the nucleic acid encapsulated in the capsid can be calculated from the measured amount of change in the ionic current Ip.

[0078] <Example 3> The ionic current of each capsid was measured in the same procedure as in Example 2 (viscosity is 1 mPa·s), except that glycerol was added to the first electrolyte and the second electrolyte so that the concentration was 30 vol% (viscosity is about 3.6 mPa·s), and the concentration of NaCl was 0.96 M.

[0079] Figure 9A is a graph showing the measurement results obtained in Example 2 (without glycerol addition), and Figure 9B is a graph showing the measurement results obtained in Example 3 (with glycerol addition). As is clear from Figures 9A and 9B, by increasing the viscosities of the first electrolyte and the second electrolyte, the discrimination accuracy of each capsid containing nucleic acids of different sizes was improved. In the example without glycerol addition shown in Figure 9A, the separation degree between an empty capsid without nucleic acid (AAV9 empty) and a capsid containing 2,599-base DNA (AAV9, 2.6 kb ssDNA) was 0.4, while the separation degree in the example with glycerol addition shown in Figure 9B was 1.13. From the above results, it was confirmed that by adding a substance with a higher viscosity than water to the first electrolyte and the second electrolyte, the discrimination accuracy of capsids containing nucleic acids of different sizes is improved.

Industrial Applicability

[0080] By the acquisition method disclosed in the present application, information regarding the size of the nucleic acid encapsulated in the capsid can be obtained. Therefore, since it can also be used for quality control of virus preparations and the like, it is useful for the medical industry.

Explanation of Reference Numerals

[0081] 1, 1a... Device for measuring ionic current, 2... Substrate, 3... Nanopore, 5... First chamber, 6... Second chamber, 7... Ammeter, 8... Analysis unit, 9... Display unit, 10... Program memory, 11... Control unit, 21... First surface, 22... Second surface, 31... First opening, 32... Second opening, 51... First chamber member, 52... First electrode, 53... Lead, 54... Power supply, 55... Ground, 61... Second chamber member, 62... Second electrode, 63... Lead, 64... Ground, S... Sample

Claims

1. A method for obtaining information related to the size of nucleic acid contained in a virus-derived capsid using a nanopore device, comprising: The nanopore device includes: A substrate having a first surface and a second surface; A nanopore penetrating from the first surface toward the second surface through which the capsid passes; A first chamber member; A second chamber member; The first chamber member forms a first chamber filled with a first electrolyte solution with at least the surface of the first opening of the nanopore on the first surface; The second chamber member forms a second chamber filled with a second electrolyte solution with at least the surface of the second opening of the nanopore on the second surface; The obtaining method includes: A capsid passing step in which the capsid contained in the first electrolyte solution or the second electrolyte solution passes through the nanopore; An ion current measuring step of measuring a change in ion current when the capsid passes through the nanopore; The capsid passing step includes: By applying a voltage to the first electrolyte solution filled in the first chamber and the second electrolyte solution filled in the second chamber, The capsid contained in the first chamber passes through the nanopore in the direction of the second chamber, or the capsid contained in the second chamber passes through the nanopore in the direction of the first chamber. Obtaining method.

2. The larger the size of the nucleic acid encapsulated by the capsid, the larger the size of the capsid The obtaining method according to claim 1.

3. The measurement step is followed by an analysis step, The analysis step analyzes the presence or absence of nucleic acid to be encapsulated in the capsid based on the change amount of the ion current measured in the measurement step. The obtaining method according to claim 2.

4. The measurement step is followed by an analysis step, The analysis step calculates the size of the nucleic acid encapsulated in the capsid based on the change amount of the ion current measured in the measurement step. The obtaining method according to claim 2.

5. The thickness of the substrate is larger than the size of the capsid. The obtaining method according to any one of claims 1 to 4.

6. The size of the nanopore is 1.2 times or more the average particle diameter of the capsid. The obtaining method according to any one of claims 1 to 4.

7. A substance having a higher viscosity than water is added to the first electrolyte solution and / or the second electrolyte solution. The obtaining method according to any one of claims 1 to 4.

8. ​ ​ A nanopore device used in an apparatus for acquiring information related to the size of nucleic acid contained in a virus-derived capsid, the nanopore device comprising: a substrate having a first surface and a second surface; a nanopore that penetrates from the first surface toward the second surface and through which the capsid passes; a first chamber member; a second chamber member; wherein the first chamber member forms a first chamber filled with a first electrolyte solution with a surface of the first surface including at least a first opening of the nanopore; the second chamber member forms a second chamber filled with a second electrolyte solution with a surface of the second surface including at least a second opening of the nanopore; the thickness of the substrate is greater than the size of the capsid nanopore device. **Claim 9** The size of the nanopore is 1.2 times or more the average particle diameter of the capsid The nanopore device according to claim 8. **Claim 10** An apparatus for acquiring information related to the size of nucleic acid contained in a virus-derived capsid, the acquisition apparatus comprising: a nanopore device; a measurement unit; an analysis unit; wherein the nanopore device comprises: a substrate having a first surface and a second surface; a nanopore that penetrates from the first surface toward the second surface and through which the capsid passes; a first chamber member; a second chamber member; wherein the first chamber member forms a first chamber filled with a first electrolyte solution with a surface of the first surface including at least a first opening of the nanopore; the second chamber member forms a second chamber filled with a second electrolyte solution with a surface of the second surface including at least a second opening of the nanopore; the measurement unit measures a change in ionic current when the capsid passes through the nanopore; the analysis unit, based on the amount of change in the ionic current measured by the measurement unit, analyzes the presence or absence of nucleic acid to be encapsulated in the capsid and / or calculates the size of the nucleic acid encapsulated in the capsid acquisition apparatus. **Claim 11** The nanopore device is the nanopore device according to claim 8 or 9 The acquisition apparatus according to claim 10.

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